p27Kip1 Immunostaining for the Differential Diagnosis of Small B-Cell Neoplasms in Trephine Biopsies Marcus Kremer, M.D., Stephan Dirnhofer, M.D., Anna Nickl, Heinz Hoefler, M.D., Leticia Quintanilla-Martínez, M.D., Falko Fend, M.D. Institute of Pathology (MK, HH, FF), Technical University Munich, Germany; Institute of Pathology (SD), University of Innsbruck, Austria; Institute of Pathology (MK, AN, HH, LQM), GSF-National Research Center for Environment and Health, Neuherberg, Germany; and Institute of Pathology (SD), University of Basel, Switzerland.

MCL were p27kip1-negative in the tumor cells, The distinction between mantle cell whereas four cases revealed a weak nuclear positiv- (MCL) and other small B-cell non-Hodgkin lympho- ity. Seventeen (77%) cases of hairy cell mas (NHL) is important because MCL has a more were also either completely negative for p27kip1 or aggressive clinical course. In bone marrow (BM) showed a faint positive in a minority of the biopsy specimens, this distinction can be particu- neoplastic cells. Nine of 19 cases (47%) of MCL larly difficult. Although cyclin D1 immunostaining showed a bcl1 rearrangement involving the major and molecular detection of the t(11;14) transloca- translocation cluster region. These findings demon- tion are highly specific markers for MCL, they fail to strate that p27kip1 immunostaining is a valuable detect a proportion of cases. We have recently de- additional marker for the differential diagnosis of scribed that MCL typically lacks detectable expres- small B-cell NHL infiltrates in BM biopsies. The sion of the cyclin-dependent kinase inhibitor kip1 kip1 reduction or lack of p27 protein expression in p27 protein by immunostaining, which is ex- MCL, as well as in hairy cell leukemia, might be an pressed at high levels in most small B-cell NHL important event in the pathogenesis of these inversely correlated to the proliferation rate. We disorders. therefore examined whether p27kip1 immunostain- ing could be a useful adjunct for the differential KEY WORDS: p27kip1 protein, Bone marrow infil- diagnosis of small B-cell NHL infiltrates in the BM. trates, Differential diagnosis, Hairy cell leukemia, Trephine BM biopsy specimens of 96 patients, in- Immunohistochemistry, , cluding well-characterized MCL (19 cases), B-cell Small B-cell non-. chronic lymphocytic leukemia (27 cases), follicular Mod Pathol 2001;14(10):1022–1029 lymphoma (18 cases), hairy cell leukemia (22 cases), and lymphoma (10 cases) as well as Morphologic examination of trephine bone marrow 10 reactive BM, including five with benign lymphoid (BM) biopsies is a standard method for staging and aggregates were investigated. In addition, the pres- follow-up of patients with malignant lymphoma (1– ence of a t(11;14) translocation involving the major 6). The incidence of BM involvement is highest in translocation cluster was studied by PCR in all MCL. small B-cell , which also make up the All cases of B-cell chronic lymphocytic leukemia, , and marginal zone lymphoma majority of cases where a primary diagnosis of non- revealed a strong p27kip1 nuclear staining in the Hodgkin lymphoma (NHL) is made on a BM biopsy majority of neoplastic cells. Fourteen (78%) cases of (4, 5). Small B-cell NHL are a diverse group of clinically, phenotypically and genotypically well de- fined disease entities (7). Despite this fact, a definite Copyright © 2001 by The United States and Canadian Academy of separation of these lymphoma subtypes on BM tre- Pathology, Inc. phine biopsies remains difficult, due to overlapping VOL. 14, NO. 10, P. 1022, 2001 Printed in the U.S.A. Date of acceptance: May 25, 2001. cytologic features and patterns of distribution of This work was in part supported by a grant from the Wilhelm Sander- the neoplastic infiltrates (2). From this group of Stiftung, Germany. Authors Quintanalla-Martínez and Fend contributed equally to this work. lymphoproliferations with a predominance of small Address reprint requests to: Falko Fend, M.D., Institute of Pathology, Technical University of Munich, Ismaninger Strasse 22, D-81675 cells, mantle cell lymphoma (MCL) stands out as a Muenchen, Germany; e-mail: [email protected]; fax: 49-89-4140-6106. more aggressive neoplasm with poor response to

1022 conventional therapeutic regimens and a median sections, in addition to standard morphologic cri- survival duration of 3 to 4 years (8, 9). Like other teria and paraffin immunostaining including small B-cell NHL, the majority of patients present monoclonal antibody DBA 44 (28–30). Cases of B- with advanced disease, including generalized CLL/SLL had been immunophenotyped by flow cy- and BM involvement (8, 10–12). tometry of peripheral blood or BM samples. Therefore, the distinction of MCL from other B-cell BM samples were formaldehyde (4%) fixed (pH neoplasms in BM biopsies is of significant clinical 7.4) for at least 24 hours and decalcified with buff- relevance. Immunohistochemical demonstration of ered sodium-ethylenediaminetetra-acetic acid (pH cyclin D1 protein is an important marker for the 7.0) for 48 hours. Four to five ␮m thick sections diagnosis of MCL, because it is expressed neither in were cut, stained with hematoxylin and eosin, Gi- normal nor in most B-cell NHL (6, 8, emsa, periodic acid-Schiff, Gomori’s reticulin stain, 13, 14). However, cyclin D1 staining can be capri- and Naphtol AS-D chloroacetate esterase. cious in decalcified BM biopsies, and false negative results may lead to misclassification of lymphoma infiltrates (6, 15, 16). Immunohistochemistry p27kip1 is a cyclin-dependent kinase inhibitor which is crucial for cell cycle progression from G1 All cases were stained for cyclin D1 (clone into S phase (17, 18). Deregulation of p27Kip1 ex- P2D11F11, Novocastra, Newcastle, UK; dilution pression is a relatively common feature in many 1:10), CD20 (DAKO, Copenhagen, Denmark; dilu- solid tumors, but alterations of the gene are infre- tion 1:500) and polyclonal CD3 (DAKO; dilution Kip1 quent (19–22). We and others have recently ob- 1:200). The expression of p27 was investigated served that the lack or reduction of p27Kip1 protein with the monoclonal antibody Kip-1 (Transduction expression is a characteristic feature of MCL, in Laboratories, Lexington, KY; dilution 1:1000). More- contrast to other small B-cell NHL, in which p27Kip1 over, in selected cases stains for CD10 (Novocastra; is strongly expressed (23–25). We therefore investi- dilution 1:10), CD5 (clone 4C7, Novocastra; dilution gated the utility of p27Kip1 immunostaining for the 1:50), CD23 (Novocastra; dilution 1:50), and MiB-1 differential diagnosis of small B-cell NHL infiltrates (Dianova, Hamburg, Germany) were accomplished. in routinely processed BM biopsies. Immunohistochemistry was performed on an au- tomated immunostainer (Ventana Medical Sys- tems, Tucson, AZ) according to the manufacturer’s MATERIALS AND METHODS protocols, with minor modifications (24). After deparaffinization and rehydration, the slides were Tissue Samples placed in a microwave pressure cooker in 0.01 Trephine BM biopsy specimens of 96 patients mol/L citrate buffer (pH 6.0) containing 0.1% were selected from the files of the Institutes of Tween 20 and heated in a microwave oven at max- Pathology of the University of Innsbruck, Austria, imum power (800W) for 35 minutes. The sections the Technical University of Munich, Germany, and were immediately cooled in Tris-buffered saline the General Hospital Salzburg, Austria. All cases and washed in 3% goat serum for 20 minutes. In- were classified according to the Revised European- cubations with the primary antibodies were per- American Lymphoma and the upcoming WHO formed overnight at room temperature. The rest of classifications (7, 26). They included 19 cases of the procedure was completed on the Ventana MCL, of which 18 were classified as typical MCL immunostainer. and 1 as blastic variant of MCL (27), 27 cases of Positive controls for all investigated antibodies B-cell chronic lymphocytic leukemia (B-CLL)/small were used to confirm the adequacy of the staining. lymphocytic leukemia (SLL), 18 cases of follicular The staining quality of cyclin D1 was verified by a lymphoma (FL) (grades I and II), 22 cases of hairy cyclin D1-positive MCL, carrying a t(11;14) translo- cell leukemia (HCL), and 10 cases of marginal zone cation. Furthermore, scattered positive endothelial lymphoma (MZL). As controls, 10 BM biopsies with cells served as internal control. For the comparison reactive changes, including five BM biopsies with of p27Kip1 staining between tissue samples, T-cells reactive nodular lymphoid infiltrates, partially with were used as internal controls. germinal centers were analyzed. Additional lymph Double immunostaining was also performed on node (LN) biopsies were investigated in five cases of representative cases to more precisely evaluate the MCL. coexpression of p27Kip1 with B- (CD20) and T- The primary diagnosis of MCL, FL, and MZL had (CD3) cell markers. For these reactions, p27Kip1 was been made on LN biopsies or extranodal tumor detected as described above followed by 2 hours of infiltrates thoroughly characterized by paraffin sec- incubation with CD20 or CD3 antibodies, respec- tion immunostaining. For a diagnosis of HCL, ex- tively. The second reaction was developed using the pression of CD103 was demonstrated on BM frozen Elite-ABC-Kit and VIP substrate (Vector laborato-

p27Kip1 in Bone Marrow Biopsies (M. Kremer et al.) 1023 FIGURE 1. p27Kip1 expression in small B-cell NHL infiltrates in the BM. A–D, BM with prominent infiltrate of a typical MCL. A, H&E; ϫ200. B, The neoplastic cells are predominantly CD20-positive B-cells. (Immunoperoxidase); ϫ300. C, CD3 staining reveals a considerable number of infiltrating CD3-positive T-cells. (Immunoperoxidase); ϫ300. D, p27Kip1 staining. The neoplastic cells show the characteristic lack of p27Kip1 expression, whereas the intermingled T-lymphocytes are strongly positive. (Immunoperoxidase); ϫ200. Insert: double staining for p27Kip1 (brown) and CD3 (purple) in the same infiltrate of MCL demonstrates that the p27Kip1-positive cells within the tumor co-express CD3. (Immunoperoxidase); ϫ600. E–G, CD20 and p27Kip1 expression in a case of B-CLL in the BM. E, H&E: ϫ200. F, The majority of neoplastic B-cells express membranous CD20. (Immunoperoxidase); ϫ300. G, Strong nuclear p27Kip1 expression in nearly all tumor cells, similar to the intensity seen in T-lymphocytes. (Immunoperoxidase); ϫ200. H–J, BM with a typical infiltrate of HCL. H, H&E: ϫ200. I, The hairy cells show a characteristic strong membranous CD20 positivity. (Immunoperoxidase); ϫ300. J, The neoplastic hairy cells are completely negative for p27Kip1, whereas some reactive T-lymphocytes and plasma cells are positive. (Immunoperoxidase); ϫ200.

1024 Modern Pathology ries, Burlingame, CA, USA) resulting in a contrast- p27Kip1 and Cyclin D1 Protein Expression in MCL ing dark purple precipitate. Fourteen of the 18 cases (78%) of classical MCL All immunohistochemical stainings were re- were classified as negative for p27Kip1, showing ei- viewed by three pathologists (MK, LQ-M, FF). Be- ther a faint reactivity of tumor cells in two cases cause cyclin D1 expression is undetectable in nor- (15%) or complete lack of staining in 12 cases (85%). mal lymphatic cells, all unequivocal, nuclear In all cases, scattered small lymphocytes strongly staining in tumor cells was classified as positive. For Kip1 positive for p27 were identified. Serial sections p27Kip1 protein expression, the staining intensity stained for CD3, as well as double stainings re- was graded as negative, weak, moderate and strong; vealed that these p27Kip1-positive small lympho- strong staining was defined as being of comparable cytes were reactive T cells (Fig. 1, A–D). In the intensity to reactive T-cells and plasma cells. A lym- Kip1 remaining four cases, the majority of the neoplastic phoma was considered as p27 -positive, if at Kip1 least 20% of the neoplastic cells showed any nuclear cells were weakly p27 -positive, at a significantly staining. lower intensity than the scattered T cells. The single case of blastic variant of MCL weakly expressed p27Kip1 in most tumor cells despite a high prolifer- ation index as assessed by MiB1 staining (more Detection of bcl1 Translocations by PCR Analysis than 80%). Nuclear cyclin D1 expression was dem- onstrated in all but two cases (89%). However, the In all MCL cases and in the HCL cases showing staining was weak and heterogeneous in many BM positivity for cyclin D1, the presence of transloca- biopsies, making interpretation problematical. tions involving the bcl1/JH major translocation cluster (MTC) was assessed by PCR using a previ- Both MCL cases negative for cyclin D1 in the BM ously published protocol (31). Briefly, DNA was ex- were cyclin D1 positive in the diagnostic LN biopsy tracted from paraffin-embedded BM and LN biopsy examined in parallel. In addition, one of these two specimens. The tissue was dewaxed, and digested cases showed a bcl1 rearrangement by PCR (Table Kip1 by overnight proteinase K incubation (20 mg/mL) 1). Both cases were p27 -negative in the BM, at 55°C. Amplification was performed using a con- supporting a diagnosis of MCL. sensus JH primer 5'-ACC TGA GGA GAC GGT GAC CAG GGT-3' and one of two MTC region primers, MTC1 5'-CCT CTC TCC AAA TTC CTG-3' and MTC2 5'-GAT GGG CTT CTC TCA CCT ACT A-3'. Forty TABLE 1. Results of Cyclin D1 and p27Kip1 Immunostaining and bcl1 Rearrangement Analysis in 19 cycles of amplification under previously estab- Cases of MCL lished, optimized conditions were performed. The Cyclin Case Tissue p27Kip1 bcl1 Rearrangement product was electrophoresed in a 2% agarose gel, D1 stained with ethidium bromide, and photographed 1a BM ϩϩϩ Ϫ ϩ under ultraviolet light. Selected positive cases were LN ϩϩϩ Ϫ ϩ analyzed by direct sequencing to confirm the spec- 2a BM ϩϪ Ϫ ificity of the amplification product. Stringent labo- LN ϩϪ ϩ 3a BM ϪϪ ϩ ratory protocols were followed to prevent PCR LN ϩϩ Ϫ Ϫ product contamination. A well characterized MCL 4a BM ϪϪ Ϫ served as positive control, and was included in ev- LN ϩϩ Ϫ Ϫ 5BM ϩϪ ϩ ery run. 6BM ϩϩ ϩ ϩ 7BM ϩϩ ϩ ϩ 8BM ϩϩ ϩ 9BM ϩϪ ϩ RESULTS 10 BM ϩϪ Ϫ 11 BM ϩϪ Ϫ ϩϩ Ϫ Ϫ Immunohistochemical Findings 12 BM 13 BM ϩϪ Ϫ P27Kip1 Protein Expression in Reactive BM 14 BM ϩϪ Ϫ 15 BM ϩϩ Ϫ Samples 16 BM ϩϪ Ϫ In normal BM, p27Kip1 protein was expressed in 17 BM ϩϩϩ Ϫ ϩ 18 BM ϩϩϩ Ϫ Ϫ the nuclei of most megakaryocytes, as well as in 19b BM ϩϩϩ ϩ Ϫ endothelial cells, plasma cells and small reactive LN ϩϩϩ ϩ Ϫ

T-lymphocytes. All other hematopoietic cells were a Four cases of MCL with primary diagnostic lymph node biopsy. Kip1 negative for p27 (32). Reactive nodular lym- b MCL blastic variant, with primary diagnostic lymph node biopsy. phoid infiltrates showed strong p27Kip1 protein ex- For cyclin D1 immunostaining: ϩ, some tumor cells positive; ϩϩ, the majority of tumor cells weakly positive; ϩϩϩ, all tumor cells strongly pression, whereas germinal centers where negative positive. Kip1 for p27 . MCL, mantle cell lymphoma; BM, bone marrow; LN, lymph node.

p27Kip1 in Bone Marrow Biopsies (M. Kremer et al.) 1025 p27Kip1 and Cyclin D1 Protein Expression in NHLs DISCUSSION Other than MCL Our study demonstrates that immunohistochem- A total of 77 cases of B-cell NHLs other than MCL ical detection of p27Kip1 protein is a valuable were also immunostained for p27Kip1 and cyclin D1. marker for the differential diagnosis of B-cell NHL The results are summarized in Table 2. All cases of infiltrates in BM biopsies. The consistent lack of B-CLL (27 cases), FL (18 cases), and MZL (10 cases) p27Kip1 protein expression allows the distinction of revealed a strong p27Kip1 nuclear staining in the MCL from other small B-cell NHL, which constantly vast majority of tumor cells, similar to the intensity show a strong p27Kip1 reactivity with the notable seen in T lymphocytes (Fig. 1, E–G). Occasional exception of HCL. However, p27Kip1 protein expres- transformed cells in FLs and B-CLLs were p27Kip1- sion does not discriminate between reactive and negative. Cyclin D1 protein was undetectable in all neoplastic lymphoid nodular infiltrates in the BM, examined cases of these lymphoma subtypes. with the exception of MCL. HCL represented a distinct group set apart from The classification of small B-cell lymphomas in the other small B-cell NHL. Five of 22 cases (23%) showed a weak to moderate p27Kip1 reactivity in the BM is a common diagnostic problem resulting more than 50% of the neoplastic cells, but less from overlapping morphologic features (1, 3, 6, 15). strong than plasma cells and scattered T lympho- The distinction of MCL from other entities is clini- cytes (Figure 1, H–J). Seventeen cases (77%) of HCL cally important because of its poor response to were classified as negative for p27Kip1, showing ei- therapy and its unfavorable prognosis (8). Since of ther a faint reactivity in rare cells (eight cases), or the description of cyclin D1 overexpression as a complete lack of staining in neoplastic cells (nine consistent feature of MCL and the availability of cases) (Table 2). The proliferation activity of neo- antibodies suitable for paraffin-embedded tissue, plastic cells was very low, as assessed by immuno- immunostaining for cyclin D1 has become an im- staining for MiB1. portant adjunct for the diagnosis of MCL (8, 13, 15, Cyclin D1 protein expression was detectable in 33). Nevertheless, immunohistochemical demon- nine cases (41%). Three cases (13%) showed a weak stration of cyclin D1 can be capricious, especially in to moderate nuclear staining in the majority of the routinely processed and decalcified BM biopsy tumor cells, whereas six cases (27%) revealed a specimens, and a negative result does not rule out a mostly weak nuclear staining in a minority of the diagnosis of MCL (6, 16). Even in well fixed and neoplastic cells. paraffin-embedded LN biopsies, immunohisto- chemistry fails to detect cyclin D1 protein in 10 to Molecular Analysis 30% of MCL (6, 15). In addition, the choice of fixa- PCR amplification of DNA extracted from tissues tive and decalcification procedure for BM biopsies of the 19 MCL cases revealed strong bands of ap- is critical for cyclin D1 staining. Fixation in a 1% propriate size in 9 (47%) of 19 cases, confirming the formaldehyde solution containing 0.4% glutaralde- presence of a bcl1 translocation involving the MTC hyde, a BM fixative widely used in Germany, com- (Fig. 2). In Cases 2 and 3 (Table 1), PCR failed to pletely abolishes staining for cyclin D1 (data not detect the bcl1 rearrangement in the BM and LN, shown). Although molecular demonstration of a respectively, irrespective of a reproducible positive bcl1 rearrangement can resolve some of the cases, signal in the additionally investigated tissues from PCR and even Southern blot analysis fail to identify the same patients. This discrepancy is most proba- a significant number of cases due to the wide dis- bly due to insufficient DNA quality in the formalin- tribution of breakpoints outside the MTC (34). Am- fixed samples, because the expected product sizes plification of the MTC by PCR renders a positive are between 370 and 600 base pairs. As expected, result only in 33 to 50% of MCL cases in paraffin- the investigated nine HCL failed to show a bcl1 embedded specimens (31, 35–37). Our detection translocation. rate of 47% is consistent with these published data.

TABLE 2. Summary of the Expression of p27Kip1 and Cyclin D1 in 96 NHL

cases p27Kip1 Cyclin D1

MCL 18 4a (22%) 16 (89%) BI.MCL 1 1a 1 (100%) B-CLL 27 27 (100%) 0 FL 18 18 (100%) 0 HCL 22 5a (23%) 9 (41%) MZL 10 10 (100%) 0

a Weak to moderate positivity in the majority of tumor cells. All other lymphoma subtypes showed strong positivity. NHL, non-Hodgkin lymphoma; MCL, mantle cell lymphoma; BI.MCL, mantle cell lymphoma blastic variant; B-CLL, B-cell chronic lymphocytic leukemia; FL, follicular lymphoma; HCL, hairy cell leukemia; MZL, marginal zone lymphoma.

1026 Modern Pathology in MCL is not due to gross rearrangements or de- letion of the p27Kip1 gene, and the mRNA levels of p27Kip1 are in a normal range (24, 25). Recently, it has been proposed that an increased degradation of p27Kip1 protein via the proteasome pathway might be an explanation for this phenomenon (25). Unexpectedly and similar to MCL, HCL lacks p27Kip1 expression irrespective of a generally very low proliferation rate. In our study, the majority of HCL cases (77%) showed low or undetectable levels of p27Kip1, and in the remaining positive cases the intensity of immunostaining was less than in the reactive T- and plasma cells. This finding confirms a recently published study by Chilosi et al., who investigated 58 HCL and found a lack of p27Kip1 protein expression in nearly all of them (93%) (38). The reason for the lack of p27Kip1 protein expres- sion in HCL is unclear. In contrast to MCL and Kip1 FIGURE 2. Detection of bcl1 translocations involving the MTC region epithelial neoplasms, no increased p27 degra- in BM biopsies by PCR analysis. A, PCR amplification with the MTC-1 dation was observed (25, 38). A common feature of primer. MW: fragment size marker; C: MCL with a bcl1 translocation both MCL and HCL is cyclin D1 protein overexpres- confirmed by sequencing as positive control; N: negative control. Lanes 1–4 show bands of appropriate size for Cases 1, 3, 5, and 6, whereas sion. There is a considerable variation in the rate of Lanes 5–7 show negative results for Cases 10–12. B, PCR amplification cyclin D1 positivity reported for HCL, ranging from with the MTC-2 primer of the same cases as shown in A; Lanes 1–4 as little as 6% to 100% in different studies (6, 14, 39, (Cases 1, 3, 5, and 6) show strong bands of the expected size. Cases 10– 12 were also negative, as shown for the MTC-1 region. 40). These results are probably due to the use of different antibodies and antigen retrieval tech- niques. Our rate of 41% positivity for cyclin D1 is in Keeping these diagnostic difficulties in mind, we accordance with these data. In contrast to MCL, decided to analyze whether the characteristic lack overexpression of cyclin D1 in HCL is not due to of p27Kip1 immunostaining in MCL can be exploited alterations involving the 11q13 region (39, 41, 42). for the differential diagnosis of BM biopsies in- Whether the overexpression of cyclin D1 in these volved in lymphoma (24, 25). two otherwise distinct B-cell neoplasms could hint In accordance with our previous study, a lack or to common mechanisms responsible for the ab- significant reduction of p27Kip1 immunostaining in sence or reduction of immunodetectable p27Kip1 a CD5-positive small B-cell neoplasia is highly sug- protein remains to be determined. In terms of dif- gestive of MCL, whereas a strong, homogeneous ferentiation between MCL and HCL, the common expression practically rules out this diagnosis (24). lack of p27kip1 expression should not pose diagnos- The usefulness of additional p27Kip1 staining for a tic problems, due to the characteristic morphology diagnosis of MCL is documented by two of our and CD5 negativity of HCL. cases, which were negative for cyclin D1 in the BM. In conclusion, our findings demonstrate that the In both of them, the diagnosis of MCL was con- consistent lack of p27kip1 protein expression allows firmed by a positive cyclin D1 staining in the addi- the distinction of MCL from other small B-cell NHL, tionally investigated primary LN biopsy. Both of making p27kip1 immunostaining a valuable addi- them showed a negative p27Kip1 staining in the BM, tional marker for the differential diagnosis of small underlining the value of this antibody for difficult B-cell NHL infiltrates in BM biopsies. cases. However, the strong positivity of reactive T-cells has to be taken into consideration for a correct interpretation of p27Kip1 stains. For small Acknowledgments: The authors thank Birgit Geist, B-cell NHL infiltrates with a high number of infil- Jaqueline Mueller, and Elonore Samson for their trating reactive T-lymphocytes, careful comparison expert technical assistance, and Dr. Otto Dietze with an adjacent section stained for T-cells is (General Hospital, Salzburg, Austria) for providing necessary. us with excellent archive material. Whereas MCL characteristically shows absence or significant reduction of immunodetectable p27Kip1, with the exception of the blastic variant of REFERENCES MCL, small B-cell NHL show high expression of 1. Thiele J, Langohr J, Skorupka M, Fischer R. Reticulin fiber Kip1 p27 in strictly inverse correlation to the prolif- content of bone marrow infiltrates of malignant non- eration rate (23–25). The absence of p27Kip1 protein Hodgkin’s lymphomas (B-cell type, low malignancy): A mor-

p27Kip1 in Bone Marrow Biopsies (M. Kremer et al.) 1027 phometric evaluation before and after therapy. Virchows 20. Loda M, Cukor B, Tam SW, Lavin P, Fiorentino M, Draetta Arch A Pathol Anat Histopathol 1990;417:485–92. GF, et al. Increased proteasome-dependent degradation of 2. Schmid C, Isaacson PG. Bone marrow trephine biopsy in the cyclin dependent kinase inhibitor p27 in aggressive colo- lymphoproliferative disease. J Clin Pathol 1992;45:745–50. rectal carcinomas. Nature Med 1997;3:231–4. 3. Coad JE, Olson DJ, Christensen DR, Lander TA, Chibbar R, 21. Tan P, Cady B, Wanner M, Worland P, Cukor B, Magi-Galuzzi McClennen RC, et al. Correlation of PCR-detected clonal C, et al. The cell cycle inhibitor p27 is an independent gene rearrangements with bone marrow morphology in pa- prognostic marker in small (T1a, b) invasive breast carcino- tients with B-lineage lymphomas. Am J Surg Pathol 1997;21: mas. Cancer Res 1997;57:1259–63. 1047–56. 22. Yang RM, Naitoh J, Murphy M, Wang HJ, Phillipson J, de 4. Crotty PL, Smith BR, Tallini G. Morphologic, immunophe- Kernion JB, et al. Low p27 expression predicts poor disease- notypic, and molecular evaluation of bone marrow involve- free survival in patients with prostate cancer. Urology 1998; ment in non-Hodgkin’s lymphoma. Diagn Mol Pathol 1998; 159:941–5. 7:90–5. 23. Sanchez-Beato M, Saez AI, Martinez-Montero JC, Mateo MS, 5. Brunning RD, McKenna RW. Tumors of the bone marrow. Sanchez-Verde L, Villuendas R, et al. Cyclin-dependent ki- In: Rosai J, Sobin LH, editors. Atlas of Tumor Pathology. 3rd nase inhibitor p27kip1 in lymphoid tissue: p27 kip1 expres- series, vol 9. Washington, DC: Armed Forces Institute of sion is inversely proportional to the proliferative index. Am J Pathology, 1994: 255–92. Pathol 1997;151:151–60. 6. Vasef MA, Medeiros LJ, Koo C, McCourty A, Brynes RK. 24. Quintanilla-Martínez L, Thieblemont C, Fend F, Kumar S, Cyclin D1 immunohistochemical staining is useful in distin- Pinyol M, Campo E, et al. Mantle cell lymphomas lack ex- guishing mantle cell lymphoma from other low-grade B-cell pression of p27/kip1, a cyclin-dependent kinase inhibitor. neoplasms in bone marrow. Am J Clin Pathol Am J Pathol 1998;153:175–82. 1997;108:302–7. 25. Chiarle R, Budel LM, Skolnik J, Frizzera G, Chilosi M, Corato 7. Harris NL, Jaffe ES, Stein H, Banks PM, Chan JK, Cleary ML, A, et al. Increased proteasome degradation of cyclin- et al. A revised European-American classification of lym- dependent kinase inhibitor p27 is associated with a de- phoid neoplasms: a proposal from the international lym- creased overall survival in mantle cell lymphoma. Blood phoma study group. Blood 1994;84:1361–92. 2000;95:619–26. 8. Campo E, Raffeld M, Jaffe ES. Mantle-cell lymphoma. Semin 26. Jaffe ES, Harris NL, Diebold J, Muller-Hermelink HK. World Health Organization classification of neoplastic diseases of Hematol 1999;36:115–27. the hematopoietic and lymphoid tissues. A progress report. 9. Banks PM, Chan J, Cleary ML, Delsol G, De Wolff-Peeters C, Am J Clin Pathol 1999;111(1 Suppl):S8–12. Gatter K, et al. Mantle cell lymphoma. A proposal for unifi- 27. Ott G, Kalla J, Ott MM, Schryen B, Katzenberger T, Müller JG, cation of morphologic, immunologic and molecular data. et al. Blastoid variants of mantle cell lymphoma: frequent Am J Surg Pathol 1992;16:637–40. bcl-1 rearrangements at the major translocation cluster re- 10. Argatoff LH, Connors JM, Klasa RJ, Horsman DE, Gascoyne gion and tetraploid chromosome clones. Blood RD. Mantle cell lymphoma: a clinicopathologic study of 80 1997;89:1421–9. cases. Blood 1997;89:2067–78. 28. Thaler J, Dietze O, Faber V, Greil R, Gastl G, Denz H, et al. 11. Fisher RI, Dahlberg S, Nathwani BN, Banks PM, Miller TP, Monoclonal antibody B-ly7: a sensitive marker for detection Grogan TM. A clinical analysis of two of in hairy cell leukemia. Leukemia entities: mantle cell lymphoma and marginal zone lym- 1989;4:170–6. phoma (including the mucosa associated lymphoid tissue 29. Salomon-Nguyen F, Valensi F, Troussard X, Flandrin G. The and monocytoid B-cell subcategories): a Southwest Oncol- value of the monoclonal antibody, DBA.44, in the diagnosis ogy Group Study. Blood 1995;85:1075–82. of B-lymphoid disorders. Leukemia Res 1996;20:909–13. 12. Norton AJ, Matthews J, Pappa V, Shamash J, Rohatiner AZS, 30. Hounieu H, Chittal SM, Al Saati T, De Mascarel A, Sabattini Lister TA. Mantle cell lymphoma: natural history defined in E, Pileri S, et al. Hairy cell leukemia. Diagnosis of bone a serially biopsied population over a 20-year period. Ann marrow involvement in paraffin-embedded sections with Oncol 1995;6:249–53. monoclonal antibody DBA.44. Am J Clin Pathol 1992;98:26– 13. Rosenberg CL, Wong E, Petty EM, Bale AE, Tsujimoto Y, 33. Harris NL, et al. PRAD1, a candidate BCL1 oncogene: map- 31. Fan H, Gulley ML, Gascoyne RD, Horsman DE, Adomat SA, ping and expression in centrocytic lymphoma. Proc Natl Cho CG. Molecular methods for detecting t(11;14) translo- Acad Sci USA 1991;88:9638–42. cations in mantle-cell lymphomas. Diagn Mol Pathol 1998; 14. Zuckerberg LR, Yang WI, Arnold A, Harris NL. Cyclin D1 7:209–14. expression in non-Hodgkin’s lymphomas. Am J Clin Pathol 32. Taniguchi T, Endo H, Chikatsu N, Uchimaru K, Asano S, 1995;103:756–60. Fujita T, et al. Expression of p21/cip1/waf1/sdi1 and p27/ 15. Chan JKC. Immunostaining for cyclin D1 and the diagnosis kip1 cyclin-dependent kinase inhibitors during human he- of mantle cell lymphoma: is there a reliable method? Histo- matopoiesis. Blood 1999;93:4167–78. pathology 1999;34:266–8. 33. Bosch F, Jares P, Campo E, Lopez-Guillermo A, Piris MA, 16. Miller KD, Munson P, Isaacson PG. Optimizing cyclin D1 Villamor N, et al. PRAD-1/cyclin D1 gene overexpression in immunostaining of mantle cell lymphoma. Histopathology chronic lymphoproliferative disorders: a highly specific 1999;34:268–70. marker of mantle cell lymphoma. Blood 1994;84:2726–32. 17. Hirama T, Koeffler HP. Role of the cyclin-dependent kinase 34. Williams ME, Meeker TC, Swerdlow SH. Rearrangement of inhibitors in the development of cancer. Blood 1995;86:841– the chromosome 11 bcl-1 locus in centrocytic lymphoma. 54. Analysis with multiple breakpoint probes. Blood 1991;78: 18. Sherr CJ, Roberts JM. CDK inhibitors: positive and negative 493–8. regulators of G1-phase progression. Genes Dev 35. Segal GH, Maiese RL. Mantle cell lymphoma: rapid polymer- 1999;13:1501–12. ase chain reaction-based genotyping of a morphologically 19. Esposito V, De Baldi A, Luca A, Groger AM, Loda M, Gior- heterogeneous entity. Arch Pathol Lab Med 1996;120 835– dano GG, et al. Prognostic role of the cyclin-dependent 841. kinase inhibitor p27 in non-small cell lung cancer. Cancer 36. Lim LC, Segal GH, Wittwer CT. Detection of bcl-1 gene Res 1997;57:3381–5. rearrangement and B-cell clonality in mantle cell lymphoma

1028 Modern Pathology using formalin-fixed, paraffin embedded tissues. Am J Clin 40. Miranda RN, Briggs RC, Kinney MC, Veno PA, Hammer RD, Pathol 1995;104:689–95. Cousar JB. Immunohistochemical detection of cyclin D1 37. Lasota J, Franssila K, Koo CH, Miettinen M. Molecular diag- using optimized conditions is highly specific for mantle cell nosis of mantle cell lymphoma in paraffin-embedded tissue. lymphoma and hairy cell leukemia. Mod Pathol 2000;13: Mod Pathol 1996;9:361–6. 1308–14. 38. Chilosi M, Chiarle R, Lestani M, Menestrina F, Montagna L, 41. Bosch F, Campo E, Jares P, Pittaluga S, Munoz J, Nayach I, et Ambrosetti A, et al. Low expression of p27 and low prolifer- al. Increased expression of the PRAD-1/CCND1 gene in ation index do not correlate in hairy cell leukaemia. Br J hairy cell leukemia. Br J Haematol 1995;91:1025–30. Haematol 2000;111:263–71. 42. Sola B, Salaün V, Ballet JJ, Troussard X. Transcriptional and 39. de Boer CJ, Kluin-Nelemans JC, Dreef E, Kester MG, Kluin post-transcriptional mechanisms induce cyclin D1 over- PM, Schuuring E, et al. Involvement of the CCND1 gene in expression in chronic lymphoproliferative disorders. Int J hairy cell leukemia. Ann Oncol 1996;7:251–6. Cancer 1999;83:230–4.

Book Review

Fu Y-S, Wenig BM, Abemayor E, Wenig BL: making a diagnosis, they are not a substitute for Head and Neck Pathology with Clinical Cor- more detailed surgical pathology texts, atlases, relation 770 pp, Kent, United Kingdom, and fasicles. The same would be true for detailed Churchill Livingstone, 2001 ($322.00). information on surgical techniques or alternative management modalities. This text is well illustrated with numerous clini- Another excellent feature of this text is the cal photographs as well as both black and white number of references for each chapter section. and color photomicrographs. It covers almost Most are extensive and current, with several con- every condition that occurs in the head and neck taining more than 500 entries. region with the exception of cervical lymphoid A deficiency of the text is the repetitive dis- lesions. The text is site-specific, including lesions cussion of the same diseases in multiple areas of the sinonasal area and ear to oral, nasopha- without adequate cross-referencing. For exam- ryngeal, salivary gland, laryngeal, thyroid, and ple, Wegener’s granulomatosis is discussed in six neck diseases. The uniqueness of this text is the collabora- different areas with only one reference to an- tive effect of pathologists with expertise in the other area. This was frustrating for many condi- head and neck region and clinicians with surgi- tions because some site areas had a detailed cal excellence in otolaryngology. This combina- discussion with good photomicrographs, while tion of expertise results in an excellent resource other areas left the reader referring back to the for medical students, pathologists, otolaryngolo- index for additional information. gists, and others diagnosing or treating diseases As an Oral and Maxillofacial Pathologist with of the head and neck region. 30 years of experience in head and neck diseases, Each chapter typically includes a section on I learned new information in almost every chap- clinical consideration and pathology. The clini- ter. The text is a valuable reference for both cal portion discusses diagnosis and treatment, pathologists and clinicians, and I would highly often including suggested imaging, surgical tech- recommend it for the clinical-pathologic niques, and alternative therapy. The pathology correlation. section includes gross and microscopic pathol- ogy, including immunohistochemical techniques Bruce F. Barker and differential diagnoses. Although photomi- University of Missouri-Kansas City crographs are generally excellent and helpful in Kansas City, Missouri

p27Kip1 in Bone Marrow Biopsies (M. Kremer et al.) 1029